ES2528132T3 - Method for diagnosing cancers that express the HER2 receptor or its truncated variants - Google Patents

Abstract

Antibody or fragment thereof that recognizes an epitope of a truncated form of the HER2 receptor defined by SEQ ID NO: 1, said epitope being defined by a sequence included in SEQ ID NO: 2, in which the antibody or fragment thereof is suitable to distinguish the protein of SEQ ID NO: 1 from the HER2 receptor.

Description

Method for diagnosing cancers that express the HER2 receptor or its truncated variants

The present invention relates to a diagnostic method in isolated samples of cancers expressing the HER2 receptor. The invention also provides compounds for use in the early diagnosis and identification of cancer as well as for use in therapy.

Prior art

HER2 (also known as c-erbB2, ErbB2 or Neu) is a type I transmembrane protein that belongs to the family of epidermal growth factor receptors or EGFR, also known as HER1 or ErbB1. Two additional members, HER3 and HER4 complete this family. When HER1, 3 or 4 binds to an EGF-type ligand, its extracellular domain adopts the so-called "open" conformation, which allows the formation of homodimers and heterodimers. Although it does not bind any ligand, HER2 also interacts with other HER receptors bound to a ligand, due to the fact that its extracellular domain is constitutively "open".

Dimerization directed by the extracellular domain leads to the interaction of the intracellular kinase of the HER receptors and the subsequent transfosphorylation of some tyrosine residues. These phosphotyrosines act as couplers of a group of intracellular phosphotyrosine binding proteins. The interactions established in the plasma membrane are transduced to the cell nucleus by means of different signaling pathways, such as the mitogen-activated protein kinase-activated protein kinase (MAPK) pathway, the stress-activated protein kinase (JNK) pathway. ), C gamma phospholipase, etc. All these signaling circuits control the expression of genes that act in a coordinated manner to modify aspects that determine the state of the cell, such as proliferation, migration, survival and cell adhesion. Therefore, depending on the cellular context, activation of the HER receptors results in a drastic cellular response, which can range between transformation in a malignant cell and premature senescence.

In addition to the canonical signaling mode, HER receptors or fragments thereof can be endocrited and transported to the nucleus, where they can directly regulate the expression of certain genes. This fact has been observed for the specific case of HER2 (Wang et al., "Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2", Cancer Cell-2004, Vol. 6, pp. 251 261), as well as for a carboxyl terminal fragment (CTF), which consists of a truncated form of the HER4 receptor, which includes the entire cytoplasmic part (Linggi et al., “ErbB-4 s80 intracellular domain abrogates ETO2-dependent transcriptional repression "

J. Biological Chemistry-2006, Vol. 281, p. 25373-25380).

In human breast tumors, a series of terminal carboxyl or CTF fragments have often been found, which are supposed to include the transmembrane and cytoplasmic domains of HER2 (Molina et al, "NH (2) -terminal truncated HER2 protein but not full -length receptor is associated with nodal metastasis in human breast cancer ”, Clinical Cancer Research-2002, Vol. 8, pp. 347-353). It is also known that breast cancer patients who express HER2 CTFs, or what is the same, truncated forms that do not include the N-terminal end of HER2 (HER2 CTF), are more likely to develop metastases (Molina et al. 2002, cited above) and a worse prognosis than those patients who mainly express the complete form of HER2 (Saez et al., "P95HER2 predicts worse outcome in patients with HER2-positive breast cancer", Clinical Cancer Research- 2006, Vol. 12, pp. 424-431).

Therefore, it is very important to be able to detect the presence of HER2 in tumors in time and even more important to determine if it is in a complete or truncated form (CTF).

Currently, at the level of routine clinical tests, antibodies are used to detect the presence of the complete form of HER2, in order to determine the type of breast tumor in question. In the case of detecting the presence of HER2, the recommended therapy consists in administering therapeutic monoclonal antibodies, such as Genentech trastuzumab. The use of this monoclonal antibody for the treatment of cancer is described in application WO 8906692 on behalf of Genentech. WO 8906692 describes monoclonal or polyclonal antibodies directed against the extracellular region of HER2, this extracellular region corresponding to the complete outer part of the protein, which is its N-terminal end. As mentioned above, the epitopes recognized by the antibodies cited in WO 8906692 are antigenic regions of the extracellular domain of the complete HER2 form and are not included in the truncated forms or carboxyl terminal fragments of HER2. Therefore, with the antibodies and the diagnostic method described in WO 8906692 it will not be possible to detect the presence of truncated HER2 (CTF). In addition, in breast tumors in which said HER2 CTFs are expressed, antibodies such as trastuzumab are not therapeutic, since they do not recognize any epitopes. This fact explains the resistance to trastuzumab treatment observed in patients expressing truncated forms (CTF) of HER2.

Patients who express truncated forms or CTF of HER2 should be treated with alternative therapies in order to avoid the numbers of poor prognosis observed by Saez et al 2006 (cited above). In order to detect

(diagnose) and treat as soon as possible people who have tumors in which truncated forms of HER2 are expressed, it is very interesting to be able to distinguish which form of HER2 is expressed in order to act accordingly.

Anido et al., "Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation", European Molecular Biology Organization (EMBO) Journal -2006, Vol. 25, p. 3234-3244, describes that in addition to the fragment generated by the action of alpha-secretases in HER2, which results in a truncated form (CTF) known as P95 that includes the transmembrane and cytoplasmic receptor fragment, two forms are also generated truncated (CTF) through an alternative translation initiation mechanism that starts from two methionines located in the 5 'and 3' directions, respectively, of the transmembrane domain of HER2. Specifically, the methionines for alternative translation initiation correspond to methionine 611 and methionine 687 of the amino acid sequence with registration number M11730.1 of the UniGene database of the National Center for Biotechnology Information (NCBI). This document also shows that these alternative forms of the HER2 receptor (CTF) are present in breast tumors. In particular, it indicates that the most abundant correspond to the form known as CTF687, or in other words, to the protein obtained by the alternative initiation of translation that starts from methionine at position 687. Anido et al. They propose as therapy the use of HER2 tyrosine kinase activity inhibitors, such as lapatinib, in order to minimize the growth of tumors expressing these truncated forms of HER2. The tyrosine kinase inhibitors act by interacting with the C-terminal end of the HER2 receptor that is integrally present in both the complete receptor and in CTFs derived therefrom or produced by the alternative translation initiation.

The Scaltriti et al document, "Expression of p95HER2, a truncated form of the HER2 Receptor, and response to anti-HER2 therapies in breast cancer", Journal of National Cancer Institute-2007, Vol. 99, p. 628-368, represents an example of a study giving rise to an alternative methodology to detect the presence of one of the truncated forms of the HER2 receptor and lists some of the possible causes of resistance to treatment with trastuzumab (Herceptin). In particular, it emphasizes the accumulation of the p95HER2 fragment (product of HER2 proteolysis by means of alpha-secretases) and other truncated forms of the receptor, which do not have the extracellular domain recognized by Trastuzumab. Scaltriti proposes an immunofluorescence method to detect the p95HER2 fragment (proteolysis product by alpha-secretases). This new detection method can be carried out in tissue sections embedded in paraffin and fixed with formalin following clinical protocols. The new methodology arises from the observation that the truncated form p95HER2, and not the entire form of the receptor, is located both in the plasma membrane and in the cytoplasm of the cell. This method proposes to compare whether there is expression of p95HER2 by staining the cytoplasm detected with an anti-HER2 antibody that binds to the cytoplasmic domain of the receptor; and confirm these results with those of a detection with an antitokeratin antibody, a protein whose distribution has been widely used as a tool for the diagnosis of tumors. However, it is not clear whether this method effectively distinguishes those tumors that express the full form of HER2 from those that express the truncated forms.

There is a need to locate new and effective targets for diagnosis and therapy, correlated appropriately with the type of cancer in question and make it possible to treat tumors that have the worst prognosis in time with effective therapies, discarding from the beginning those therapies that have been observed that are not effective.

The present invention offers benefits related to the problems cited above and represents a novel solution in the early classification of cancer, specifically breast cancer.

Summary of the invention

It has been determined that the presence of one of the truncated forms (CTF) of HER2, generated by the alternative start of translation, correlates very well with the prediction of the type of cancer to be treated, making the doctor's task easier at the time of prescribing a treatment cycle. This truncated form of HER2 (CTF-611) has been identified and found to have the sequence SEQ ID NO: 1.

This sequence has been used to develop several tools to detect its presence in tissue samples, thus providing a solid new diagnostic set that is also applicable in clinical routine.

The present invention also provides new antibodies, or fragments thereof, that recognize this truncated form or CTF of HER2, which is not recognized by trastuzumab. These antibodies recognize an epitope that is defined by a sequence included in SEQ ID NO: 2. The invention also provides hybridoma cell lines suitable for producing such antibodies. The currently available anti-HER2 antibodies recognize CTF and HER2 or only HER2, making it very difficult to discriminate patients expressing only HER2 from patients expressing HER2 and CTF. Unlike the available anti-HER2 antibodies, the antibodies described herein preferably bind to CTF-611, allowing the identification of patients expressing this CTF.

The present invention also relates to a method of diagnosing cancer in patient samples that

it comprises the detection of the presence in said sample of the truncated form of the HER2 receptor consisting of SEQ ID NO: 1.

The present invention also provides the use of the antibody or fragment thereof of the present invention for the diagnosis and determination of prognosis in isolated samples of cancers in which the HER2 receptor is expressed.

The present invention further provides a diagnostic and prognostic determination agent in isolated samples of cancers in which the HER2 receptor and / or C-terminal fragments are expressed, comprising at least one antibody or a fragment thereof as described above. .

The present invention also provides a diagnostic and prognostic determination kit in isolated samples of cancers, in which the HER2 receptor is expressed, comprising means for detecting the presence in said sample of the truncated form of the HER2 receptor consisting of the SEQ ID NO: 1.

The invention also provides an antibody or a fragment thereof as described above for use in therapy or in the manufacture of a medicament for the treatment or prevention of cancers in which at least the truncated form of the HER2 receptor is expressed which consists of SEQ ID NO: 1.

According to another feature of the invention, the use of antibodies or fragments is for the manufacture of a medicament for treating or preventing breast cancers in mammals, including humans.

Another object of the present invention is a pharmaceutical composition for the treatment of cancers in which at least the truncated form of the HER2 receptor consisting of SEQ ID NO: 1 is expressed, which comprises an antibody or a fragment thereof as described. above and at least one pharmaceutically acceptable excipient and / or vehicle.

Brief description of the drawings

Figure 1: Diagram of the protein sequence of the truncated form CTF-611, in comparison with the complete HER2 receptor sequence and with the truncated form known as p95 (648-CTF / p95), product of proteolysis by alpha-secretases . The transmembrane domain is represented as a helical line. The rest of the molecule is indicated with a gray box. The peptide sequence used to generate mono and polyclonal antibodies is underlined. The positions of intramolecular disulfide bridges are also indicated with connections between cysteines.

Figure 2: Western-type electrophoresis and immunoblot (membrane transfer) of breast cancer samples. (S) means soluble fraction and (M) membrane fraction. In order to detect complete HER2 and the CTF-611 form, antibodies directed to the cytoplasmic domain of the proteins were used. DHL: Lactate dehydrogenase (used as a control).

Figure 3: Results of the evaluation of the number of tumors developed in transgenic mice expressing the truncated form CTF-611 (figure 3A) compared to transgenic mice expressing the complete HER2 receptor; and the results of the volume of tumors detected (Figure 3B). On the Y axis, N and V mean Number of tumors and Volume of tumors, respectively. On the X axis, T refers to Time in weeks.

Figure 4: Figure 4A shows electrophoresis and Western blotting (membrane transfer) revealed with an antibody (CB11) that recognizes the cytoplasmic domain of the HER2 receptor (common domain in all complete and truncated forms) or with polyclonal antibodies generated against the peptide of SEQ ID NO: 3 (-611-A and -611-B). The lanes correspond to lysate extracts of MCF7 cells (previously transformed with the constructs of Figure 1) that express the complete form of the HER2 receptor, the truncated form CTF-611 and the truncated form p95. Figure 4B shows the result of an immunoprecipitation test with the -611-A, -611-B and CB11 antibodies carried out on samples containing both the complete HER2 receptor and the truncated forms CTF-611 and p95.

Figure 5: (A) MCF7 cells expressing HER2, 611-CTF or 648-CTF were lysed and analyzed by Western blotting with CB11, an antibody against the cytoplasmic domain of HER2, or with two independent anti-611 monoclonal antibodies -CTF produced against the MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC peptide (see Figure 1). (B) Lysates of MCF7 cells expressing HER2, 611-CTF or 648-CTF 1: 1: 1 were mixed and subjected to immunoprecipitation with the indicated monoclonal antibodies. The washed immunoprecipitates were analyzed by Western blotting with the CB11 antibody. A simulated immunoprecipitation without antibody (-) was performed as a negative control. (C) MCF7 cells expressing HER2, 611-CTF or 648-CTF were analyzed in a confocal microscope by indirect immunofluorescence with the indicated antibodies.

Figures 6 (A) and (B): Characterization of a monoclonal antibody by flow cytometry.

Figure 7: Characterization of monoclonal antibodies by immunohistochemistry.

Figure 8: Characterization of epitopes. Figure 8 (A): Graph showing the main sequence of the juxtamembrane region of 611-CTF and the sequence of the different constructs used to map the epitopes. Figure 8 (B) Western blots.

Figure 9: Analysis of breast cancer samples. Figure 9 (A) Table with analytical results. Figure 9 (B) Immunocytochemical staining.

Detailed description

CTF-611

As mentioned earlier, the inventors found that, surprisingly, the differential detection of said truncated form of HER2 with SEQ ID NO: 1 correlates very well with the manifestation of a common type of cancer in breast tissue with a poor forecast. Therefore, Figure 3 shows the results of the evaluation of the number of tumors developed in transgenic mice expressing the truncated form CTF-611 (SEQ ID NO: 1).

In order to obtain the results of this figure 3, cDNA constructs were expressed in the mouse breast epithelium, coding said constructs for human SEQ ID NO: 1 (CTF-611), identified in the figure as M611-CTF . This construct comprises SEQ ID NO: 1 under the control of the mouse mammary tumor virus (MMTV) promoter. As a control, the FVB / N-Tg (MMTVneu) 202J mouse line expressing the complete form of HER2 was used, also under the control of the MMTV promoter. As can be deduced from Figure 3A, the number of tumors in mice expressing the cDNA construct with the form CTF-611 (SEQ ID NO: 1) is much greater than that of the FVB / N-Tg line (MMTVneu) 202J, identified in this figure with the designation HER2 / neu. Tumors were detected in the transgenic animals at 17 weeks of age of the transgenic animals expressing CTF-611, which represents three months before the first detection in FVB / NTg (MMTVneu) 202J mice. This fact would explain the greater aggressiveness of the tumors that express HER2 fragments in relation to the tumors that express the complete form. Figure 3B also shows that the volume of tumors is much higher in animals that express the truncated CTF form of HER2 with SEQ ID NO: 1, relative to animals constitutively expressing the complete receptor, also known as HER2 / neu . This second fact also explains the greater aggressiveness of this type of tumors. The inventors also determined that tumors expressing the truncated form of sequence SEQ ID NO: 1 showed higher rates of pulmonary metastasis than those observed in tumors expressing HER2 / neu. Example 9 below provides additional experiments that demonstrate clinical relevance.

All these data with transgenic animals reveal that differential detection of the truncated form of the HER2 receptor of SEQ ID NO: 1 with respect to the complete form of the receptor is very necessary.

These truncated forms of HER2 may contain neo-epitopes, in other words, new antigenic determinants not present in the complete receptor molecule, which means that they do not interact in the same way with the molecules as with the complete receptor (antibodies, medications, etc.).

Antibodies against CTF-611

The inventors were able to produce both polyclonal and monoclonal antibodies against CTF-611. These antibodies recognize an epitope that is defined by a sequence included in SEQ. ID NO: 2, preferably by a sequence included in or defined by SEQ. ID NO: 3. Most preferred are antibodies that recognize an epitope that is included in or defined by MPIWKFPDEEGAS (SEQ. ID NO: 5).

In the context of the present invention, it is understood that epitope means the part of a peptide type macromolecule (or an antigen), whose sequence and / or spatial configuration is recognized by the immune system (antibodies, T cells, B cells) .

The antibodies of the present invention can preferably distinguish the protein of SEQ ID NO: 1 from the HER2 receptor. In addition, the antibodies of the present invention can preferably distinguish the protein of SEQ ID NO: 1 from the truncated form 648-CTF / p95 of the HER2 receptor. This distinction can be visualized by one or more of immunofluorescence, flow cytometry, immunohistochemistry in cultured cells and immunohistochemistry in patient samples. It is particularly preferred that the distinction can be quantified both in flow cytometry and in immunoprecipitation.

According to the present invention, the binding of the antibodies of the present invention to 611-CTF in an immunoprecipitation experiment is preferably at least 300 times, more preferably at least 1000 times, most

preferably 1000 to 2000 times stronger than its binding to HER2, as described in more detail in example 3 (using 5 micrograms of 32H2 and 5 micrograms of 20F4),

As indicated above, the antibody can be polyclonal or monoclonal.

Within the meaning of the present invention, "polyclonal antibody" is understood to mean the group of antibodies produced by different B cell lines (B lymphocytes). Polyclonal antibodies are mixtures of immunoglobulins secreted against an antigen (macromolecule), each of these immunoglobulins being able to recognize a different epitope, since it comes from a different B cell. Therefore, within the different populations of immunoglobulins contained in a polyclonal antibody, there is a type of immunoglobulin that recognizes the epitope or epitopes of interest of a specific antigen.

Polyclonal antibodies can be produced, for example, by conjugating the peptide of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 with an immunogen such as Californian barnacle hemocyanin and immunizing rabbits with this conjugate.

Monoclonal antibodies can be produced using the same or similar immunogens. Hybridoma cell lines can be produced in a manner known to the expert. The hybridoma cell line can then be grown in a suitable culture medium from which the monoclonal antibody is recovered.

In a preferred embodiment, they are produced by the hybridoma cell line deposited in the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH -DSMZ" with registration number DSM ACC2904 according to the Budapest Treaty of April 9, 2008 or by the cell line of hybridoma deposited with the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH - DSMZ" with registration number DSM ACC2980 according to the Budapest Treaty of November 6, 2008.

Particularly preferred are monoclonal antibodies produced by these hybridoma cell lines, as well as monoclonal antibodies that are at least functionally equivalent. At least equivalent from a functional point of view are those antibodies that can discriminate between CTF-611 and HER2 equally well or even better.

Useful antibodies also include humanized and human antibodies.

Therefore, the present invention also provides isolated peptides of sequence SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO: 4 or SEQ ID NO: 5.

Instead of the complete antibody, a fragment thereof according to the present invention can be used. The antibody fragments are selected from the group comprised of F (ab), F (ab ’) and Fv. In the context of the present invention, a fragment of an antibody refers to a part of the antibody that is of sufficient size and of appropriate structure to bind an epitope present in the truncated form of the HER2 receptor and therefore to allow its detection in a sample.

Examples 2 and 3 show specific antibodies. Obviously, the invention extends to other polyclonal or monoclonal antibodies directed against the epitope of the truncated form CTF-611 which is defined by the sequences SEQ ID NO: 2 and SEQ ID NO: 3, since, based on the teachings of this invention, can be obtained directly by one skilled in the art. Likewise, the invention extends to fragments of said antibodies, such as F (ab), F (ab '), Fv, etc., or to any hybridoma cell line that can produce monoclonal antibodies directed against the peptides of SEQ ID NO: 2 and SEQ ID NO: 3.

Diagnostic methods

In the diagnostic method according to the invention, the detection of the presence of the truncated form of the HER2 receptor can be carried out through means selected independently of the group comprising: detection by differential migration in a mobile phase system - stationary phase of the truncated form of the HER2 receptor consisting of SEQ ID NO: 1 with respect to the complete form of said HER2 receptor; and detection by binding with specific antibodies of the truncated form of the HER2 receptor containing SEQ ID NO: 1.

Detection through differential migration should be understood to mean, in the context of the present invention, any analytical technique that allows the separation of the compounds to be detected based on their different mobility in a specific mobile phase-stationary phase system, such As an electrophoresis. Such different mobility can be caused by a different electrical charge in the compound, different molecular weight or different affinity for other compounds.

This differential detection can be carried out through analytical techniques known to those skilled in the art such as protein electrophoresis, molecular exclusion chromatography, affinity testing of

antibodies, immunofluorescence, immunocytochemistry, immunohistochemistry, flow cytometry, etc. Immunohistochemistry is particularly preferred. One or more of these methods can be combined to enhance reliability.

Differential detection of the truncated form of the HER2 receptor consisting of SEQ ID NO: 1, also referred to in this invention as CTF-611, with respect to the complete or total HER2 receptor, implies being able to visualize two proteins with different molecular weights and different sequences such as may be desired from Figure 1. The truncated form with SEQ ID NO: 1 consists of the protein that results from the alternative initiation of translation through methionine 611 of the complete HER2 receptor sequence. Therefore, this form or CTF comprises a new extracellular domain, a transmembrane fragment and a cytosolic domain. The transmembrane fragment and the cytosolic domain are the same as those in the complete form of the HER2 receptor. Another truncated form corresponds to that of Figure 1 called p95 or CTF-648. This last form is the product of the action of alpha-secretases in the complete HER2 receptor, which divide a large part of the extracellular domain.

According to another embodiment of the invention, the step of detecting the presence in the sample of the truncated form of the HER2 receptor consisting of SEQ ID NO: 1, comprises the detection with at least one antibody as defined above. In a particular embodiment, the diagnostic method according to the invention comprises the detection of the epitope defined by SEQ ID NO: 3.

In one embodiment of the invention, the antibodies or fragments thereof of the present invention are used as agents for diagnosis and prognostic determination, either directly labeled in their own peptide sequence (for example with radioisotopes) or indirectly ( for example by adding or binding a fluorescent agent or an agent that can produce fluorescence by reaction in a selected reaction medium), all of the above having the objective of generating a visible signal, and if possible quantifiable, with which detect the presence of the truncated form of the HER2 receptor consisting of the sequence SEQ ID NO: 1 in a sample.

In the same way, it is contemplated that the diagnostic agent containing at least the antibodies adheres to a solid support, directly or indirectly by means of a spacer arm. An agent of this type allows to capture the sequence form SEQ ID NO: 1 of a sample, in order to determine its presence later with other non-specific antibodies (such as CB11).

The diagnostic and prognostic agents according to the invention can also be applied in immunohistochemical protocols. For this purpose, monoclonal or polyclonal antibodies (primary or secondary) must be properly labeled to produce a visible signal, and in the best quantifiable cases, once they have come into contact with the tissue under test and have been able to interact with the proteins whose detection is desired, in other words, with the HER2 CTF fragment of sequence SEQ ID NO:

one.

In order to facilitate the task of analysis, diagnosis and determination of the prognosis to the doctor, it is contemplated that the diagnostic agent be provided in the form of a kit that includes, in addition to the antibodies, the reagents (for example reagents required for staining immunohistochemistry), tampons and detection solutions adapted to determine the presence of the truncated form of SEQ ID NO: 1 in a sample, possibly control slides representing different levels of CTF-611 expression and possibly also detailed instructions, which help the doctor in the diagnosis evaluation. This kit may also comprise a means for carrying out a semi-quantitative immunohistochemical assay for the determination of HER2 (such as Herceptest ™).

Therefore, the invention provides a kit for the diagnosis and determination of the prognosis, comprising at least one antibody or a fragment thereof, said antibody or fragment being able to recognize the epitope defined by SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 5, in addition to the reactive media and buffers suitable for carrying out the interaction of the antibody with the epitope and well known to those skilled in the art.

The diagnostic method of the present invention, and the kit of the present invention, can allow the prediction of lymph node metastasis, poor prognosis and resistance to Herceptin ™.

Another type of kit also object of the invention, comprises all the means necessary to carry out the detection through differential migration of the truncated form of the HER2 receptor consisting of the sequence SEQ ID NO: 1, in relation to the complete form of the HER2 receiver.

Within the group of cancers that express the HER2 receptor or its truncated variants, breast cancer stands out. Other cancers in which these proteins are also expressed include cancer of the lung, pancreas, colon, stomach, prostate, head and neck, skin, kidney, testis, thyroid, urinary bladder, uterus, vulva, endometrium, ovary, esophagus, mouth , salivary gland, larynx, peritoneum, nasopharyngeal region, fallopian tubes, Wilms tumors as well as lymphomas, Swing sarcomas, synovial sarcoma, medulloblastomas, trophoblastic tumors, gliomas,

glioblastomas, cholangiocarcinomas, cholesteatoma, chondrosarcoma, ependymoma, neurilemomas, neuromas, rhabdomyosarcomas. Therefore, the diagnostic method of the present invention is particularly suitable for diagnosing one of these cancers. In addition to the ex vivo detection of CTF, antibodies can be used for imaging in vivo.

Therapeutic methods

The antibodies of the present invention, or fragments thereof, are also useful in therapy, particularly in cancer therapy, particularly in those cancers that express the HER2 receptor or its truncated variants. Humanized and human antibodies and fragments thereof are preferred.

The antibodies of this invention are also used in pharmaceutical compositions useful for the treatment of cancers in which at least the truncated form of the HER2 receptor consisting of SEQ ID NO is expressed: 1. The composition comprises those pharmaceutically acceptable carriers or excipients and the less an antibody that recognizes the epitope defined by the sequences SEQ ID NO: 2 or SEQ ID NO: 3. The pharmaceutical composition may also comprise a mixture of antibodies that, in addition to an antibody of the present invention, contains other antibodies against HER2 or fragments thereof, such as Herceptin ™.

Based on the figures provided herein and always by way of illustrative but not limiting example, the method of diagnosis and prognosis of cancers in which the HER2 receptor and / or its terminal carboxyl fragments are expressed are described below ( truncated variants); new specific peptides and antibodies against said peptides; new cell lines; diagnostic agents and kits for detection, all of which are objects of the invention.

Although not specified, all technical and scientific terms used in the specification have the meaning that a person skilled in the art, to whom the invention belongs, would assign them. Throughout the description and the claims, the word "comprises" and variations of the word, such as "comprising", are not intended to exclude other technical characteristics, additives, components or steps. The objects, advantages and additional features of the invention will become apparent to those skilled in the art upon examination of the description or can be learned by practicing the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

In addition, it will be understood that the present invention covers all possible combinations of preferred and particular groups described above.

Examples

The following examples show different ways of detecting the presence of the truncated form of sequence SEQ ID NO: 1, in an isolated cancer sample of the type expressed by the HER2 receptor and / or its truncated variants.

Experimental procedures

Cells. MCF7 Tet-Off cells (BD bioscience) were maintained at 37 ° C and 5% CO2 in DMEM / F-12 (1: 1) (Gibco) containing 10% FBS (Gibco), 4 mM L-glutamine (PAA Laboratories ), G418 0.2 mg / ml (Gibco) and doxycycline 1 /g / ml (Sigma). Cells were transfected with the various expression plasmids using FuGENE6 (Roche). Individual stable clones with plasmids based on pUHD10-3h integrated with 0.1 mg / ml hygromycin B (Invitrogen) were selected. Expression of cDNA encoded by pUHD10-3h of HER2 and CTF was induced by removing doxycycline. First, the cells were separated with 0.5% Trypsin-EDTA (GIBCO), washed three times by centrifugation and the medium was changed 10 hours after sowing in culture plates. The homogeneity of the individual clones was checked by confocal immunofluorescence microscopy with an antibody against the cytoplasmic domain of HER2. Two independently selected stable clones were used in the experiments.

Western blot. Cells expressing the different isoforms of HER2 were lysed in modified RIPA buffer (20 mM NaH2PO4 / pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 100 mM PMSF, 25 mM NaF, 16 µg / ml aprotinin, 10 µg / ml leupeptin and 1.3 mM Na3VO4) and protein concentrations were determined with DC protein assay reagents (BIO-RAD). Samples were mixed with loading buffer (final concentrations: 62 mM Tris pH 6.8, 12% glycerol, 2.5% SDS) with 5% betamercaptoethanol and incubated at 99 ° C for 5 min before fractionation. g of protein by SDS-PAGE. Specific signals were quantified in Western blots with ImageJ 1.38 (NIH) software.

Immunoprecipitation Cell lysates were incubated with different antibodies for 1 hour at 4 ° C. Then, the immunocomplexes were purified with protein A. The immunoprecipitates were washed three times with lysis buffer, mixed with loading buffer and analyzed by Western blot.

Cells were washed for immunofluorescence microscopy seeded on glass slides with PBS, fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. PBS with 1% BSA, 0.1% saponin and 0.02% NaN3 was used for blocking and binding to the antibody, and Vectashield with DAPI (Vector laboratories) was used for the preparation.

Flow cytometry. MCF7 cells expressing HER2, 611-CTF or 648-at 4 ° C were washed with PBS and separated in PBS containing 5 mM EDTA. Separated cells were incubated with 10 µg / ml of anti-32H2 monoclonal antibody in PBS containing 5% BSA for 30 min at 4 ° C, washed and stained for 30 min at 4 ° C with FITC conjugated mouse anti-IgG antibody ( Becton-Dickinson) in PBS containing 5% BSA. Flow cytometry was performed in a FACscan using FACscan Research software (Becton Dickinson Immunocytometry Sys., Mountain View, CA).

Immunohistochemistry MCF7 cells expressing HER2, 611-CTF or 648-at 4 ° C were washed with PBS and separated in PBS containing 5 mM EDTA. Then, the cells were centrifuged and the cell pellets fixed in 10% neutral formalin, dehydrated and embedded in paraffin. Sections of the cell pellets or human tissues of a thickness of 4 µm were placed on glass slides covered with poly-lysine. Immunohistochemical analyzes were performed using the following protocol:

1. Dewax and rehydrate the cut

1.1 Incubate the slides 30 min at 60 ° C.

1.2 Dewax the slides with three 5 min incubations of transparent xylene, followed by two 3 min washes with absolute ethanol.

1.3

 Gradually bring to distilled water: Ethanol 95ºC 3 min, ethanol 70ºC 3 min, ethanol 50ºC 3 min, distilled water.

2.

 Recover antigens

PT Link at low pH (6), Envision Flex target recovery solution at high pH 10x. DM 812. 20 min at 95 ° C. Wash with wash buffer Envision Flex x10, DM 811, 15 min.

3.

 Immunohistochemical staining AUTOSTAINER more Link DAKO Envision Flex + mouse kit, high pH (Link): Envision Flex SM801 peroxidase block. Envision Flex / HRP SM 802. Envision Flex DAB + DM 807 chromogen. Envision Flex SM 803 substrate buffer. Envision Flex 10x DM 811 wash buffer. Recovery solution of Envision Flex target at high pH 10x DM 812. Envision Flex + mouse (linker) SM 84. Peroxidase 5 min and washing with wash buffer. 5% protein block, 15-20 min. Wash with wash buffer.

 32H2 diluted 1: 1000-1: 3000 (stock solution 1 mg / ml) or 20F4 diluted 1: 20-1: 50 (stock solution 1 mg / ml) 2 hours.  Wash with wash buffer.  Secondary (Flex HRP, Flex + mouse / rabbit) 20 min.

 Wash with wash buffer.

 DAB 5 min.

5  Wash with wash buffer.

Hematoxylin

 Wash with distilled water. 10

3. Dehydrate and stabilize with preparation medium

3.1 Wash gradually with increasing concentrations of ethanol (50%, 70%, 95%, 2 min each wash).

15 3.2 Gradually bring to distilled water (95% ethanol, 70%, 50%, distilled water, 3 min each wash).

3.3 Wash with xylene / eucalyptol (3 washes 2 min each).

3.4 Perform preparation with DPX.

20 transgenic mice

TG 611 and TG 687 mice were genetically engineered by cloning the sequences encoding 687-CTF and 611-CTF at the multiple cloning site II in the 3 'direction of the long terminal repeat of the mouse mammary tumor tumor virus boosted by the Rous sarcoma virus of the pMB vector (a kind gift from Dr. Marcos Malumbres, CNIO, Madrid). The founding lines were generated by microinjecting linearized plasmid DNA into fertilized oocytes collected from superovulated FVB mice at the Center for Animal Biotechnology and Gene Therapy (Center for Animal Biotechnology and Terèpia Gènica, Autonomous University of Barcelona). Founding mice were genotyped by Southern hybridization analysis. Upon identification of

30 founding animals, routine colony maintenance was performed by PCR genotyping. FVB / N-Tg (MMTVneu) 202J male and female mice were obtained from the Jackson Laboratory (Bar Harbor, ME).

Histology and complete preparations

The mammary glands were prepared on glass slides, fixed overnight in 4% paraformaldehyde and transferred to 70% ethanol. The slides were rinsed in water for 5 min and stained in a 0.2% carmine filtered solution for 24 hours. The glands were then sequentially dehydrated with decreasing concentrations of ethanol, then degreased and stored in methyl salicylate. For histological analysis, the fixed glands were blocked in paraffin, cuts were made, and stained with

40 hematoxylin and eosin.

Example 1: Detection of the fragment of SEQ ID NO: 1 through differential migration.

In Figure 2, which corresponds to an image of an electrophoresis gel and subsequent Western-type transfer

45 (Western blot), different breast cancer samples (108, 114, 101, 103, 131, 134 and 145) were loaded on the rails. From each sample, both the soluble fraction (S) and the membrane fraction (M) of the cell lysate were analyzed, in order to visualize what type of HER2 molecule was present and in which fractions. In principle, both the complete receptor and the form of SEQ ID NO: 1 are expected to be in the cell membrane. For the detection of complete HER2 and of the CTF-611 form, antibodies (CB11) were used

50 targeting the cytoplasmic domain of proteins, which is a common domain in both forms of protein. As an analysis control, the presence of the enzyme lactate dehydrogenase (DHL) was detected. In most samples, bands corresponding to the complete HER2 receptor appear and in the membrane fraction, a band corresponding to the CTF-611 form of SEQ ID NO: 1.

Therefore, Figure 2 shows that the detection of the type of truncated forms of the HER2 receptor can be carried out through a protein electrophoresis analysis. In addition, the presence of an HER2 fragment of sequence SEQ ID NO: 1 is indicative of a certain type of cancer, in the case of the example, breast cancer, which needs to be evaluated and treated as an individual case within the cancers that express HER2.

Example 2: Detection of the presence of the HER2 fragment of sequence SEQ ID NO: 1 with polyclonal antibodies directed to neo-epitopes defined by SEQ ID NO: 2 or SEQ ID NO: 3.

In order to be able to detect the presence of the truncated form of HER2, whose sequence is that described in SEQ ID NO: 1, using alternative means to the detection by differential migration in electrophoresis, a peptide having the sequence was synthesized SEQ ID NO: 4 and four rabbits were immunized with said peptide. This peptide

corresponds to the 32 amino acids of the N-terminal end of the form CTF-611 or of SEQ ID NO: 1, in which most of the cysteines have been replaced by serines in order to conjugate them with the immunogen known as lapa hemocyanin Californian (KLH).

Therefore, SEQ ID NO: 4 corresponds to a peptide equivalent to that of SEQ ID NO: 3, although adapted in order to carry out the immunization technique. One skilled in the art will understand that antibodies directed against SEQ ID NO: 4 of the synthesized peptide will also recognize the epitope defined by SEQ ID NO: 3 present in the truncated form of the HER2 receptor (SEQ ID NO: 1 or CTF-611 ). Similarly, one skilled in the art can deduce that if the synthesized peptide used for immunization consists of SEQ ID NO: 2, which comprises all the amino acids of the CTF-611 receptor located in the extracellular zone, the results with antibodies directed against This other peptide is equivalent and therefore useful for the same purpose.

SDS electrophoresis and subsequent Western-type transfer of lysate extracts from MCF7 cells (previously transformed with the constructs of Figure 1), expressing the complete HER2 receptor form, the truncated form CTF-611 and the truncated form p95. The truncated form of SEQ ID NO: 1 actually appeared as two fragments of similar molecular weight. As can be deduced from tests performed with glycosidase-F, an enzyme that eliminates protein N-glycans (not shown), the CTF-611 fragment is a substrate of post-translational modifications. Specifically, a fragment with approximately 110 kDa corresponds to the form that is synthesized and subsequently enters the secretory pathway, in which it becomes N-glycosylated.

Serum from two of the immunized rabbits recognized both the complete HER2 receptor and CTF-611. This can be seen in Figure 4A, in which the bands corresponding to both the complete HER2 receptor and the form of SEQ ID NO: 1 or CTF-611 are detected. As indicated in said Figure 4A, Western blotting was revealed with an antibody (CB11) that recognizes the cytoplasmic domain of the HER2 receptor (common domain in all forms, complete and truncated); or with the polyclonal antibodies generated against said peptide of SEQ ID NO: 4 (-611-A and -611-B) and which are present in rabbit sera. Since it is comparable to the complete HER2 signal and CTF-611, it is concluded that the -611-A and -611-B antibodies recognize a linear epitope that is present in both forms of the receptor. As a negative control in SDS electrophoresis and Western blotting, an MCF7 cell lysate was used that expressed the shape of the receptor known as p95, which does not possess the epitope against which the antibodies were designed.

In contrast to these results, when an immunoprecipitation assay was carried out with the -611-A and -611-B antibodies and the CB11 antibody, it was detected that antibodies directed against the peptide of SEQ ID NO: 4 precipitated preferably the truncated form of the receptor (CTF-611). The immunoprecipitation mixture contained a 1: 1: 1 ratio of cell lysates expressing the different forms of the HER2 receptor. These results are evident in Figure 4B. In this figure, the bands of an electrophoresis with SDS and subsequent Western-type transfer appear, in whose lanes the results of immunoprecipitation tests with the -611-A, -611-B and CB11 antibodies were loaded. A control lane (inlet) was loaded with the 1: 1: 1 mixture of the three types of cell lysates subjected to immunoprecipitation with the different antibodies. Western blotting was revealed with CB11. (Approximately 106 cells expressing full-length HER2, 611-CTF or 648-CTF were lysed in 500 µl of lysis buffer (50 mM Tris HCL pH 7.4, 137 mM NaCl, 2 mM EDTA, 10% glycerol , 1% NP40) The lysates were clarified by centrifuging at 14000 gx 30 minutes.

Input: 5 μl of lysate mixture (HER2 full length: CTF611: CTF648, 1: 1: 1)

IP: 50 µl of lysate mixture + 5 µl of rabbit anti-611 CTF rabbit or CB11 serum

In the lanes corresponding to immunoprecipitation with the -611-A and -611-B antibodies, bands can be distinguished that are clearly more intense than the rest in those molecular weights corresponding to the glycosylated and non-glycosylated form of CTF-611. That is, said antibodies directed against the peptide of SEQ ID NO: 4 did not immunoprecipitate the complete form of the HER2 receptor, which means that they actually recognize an epitope that is masked when the complete HER2 receptor is not denatured. Therefore, it can be concluded that the -611-A and -611-B antibodies are specific for CTF-611.

This specificity is corroborated by indirect immunofluorescence assays (not shown) in which affinity-purified polyclonal antibodies -611-A and -611-B only allow staining in samples expressing the truncated CTF-611 fragment. This fact implies the advantage of being able to use them to differentially detect which form of the HER2 receptor is expressed in an isolated sample of tumor tissue. Affinity purification of polyclonal antibodies was performed as follows:

(i) Purification of total IgG using a HiTrap Protein A HP column (GE Healthcare). Rabbit serum antibodies were immobilized on the equilibrated column with binding buffer (Na2HPO4), washed with 10 volumes of binding buffer column and eluted in 10 volumes of pH citric acid (2.7). Elution was neutralized using Tris-HCL pH 8.

(ii) Purification using a peptide immobilized on a HiTrap NHS column. The same peptide used to immunize the rabbit was immobilized on a HiTrap NHS column. The purified IgGs in the previous step were loaded on the equilibrated column with binding buffer (Na2HPO4), washed with 10 volumes of binding buffer column and eluted in 10 volumes of citric acid pH (2.7). Elution was neutralized using Tris-HCL pH 8.

(iii) Finally the purified antibody was dialyzed against PBS 0.02% NaN3.

The complete HER2 receptor comprises, near the cell membrane, a structured region maintained by six disulfide bridges, indicated in Figure 1 by connection lines between cysteines. The truncated form CTF-611,

or of sequence SEQ ID NO: 1, contains only five cysteines between which said disulfide bonds are established to stabilize the homodimers in this truncated form. As can be deduced from Figure 4 in its entirety, it should be concluded that the area near the cell membrane of the truncated form of SEQ ID NO: 1 is antigenically different from its equivalent in the entire HER2 receptor. It follows that it can be detected differentially according to what has been indicated above.

The quantification of the different isoforms of HER2 in the immunoprecipitates of entry, CB11 and anti-611-B normalized with respect to the number of occurrences of HER2 is as follows:

Entry

CB11 Anti -611-B

HER2

one one one

611-CTF

1.47 1.27 23.20

648-CTF

1.53 2.76 2.4

Quantification was carried out using ImageJ 1.38.

Example 3: Detection of the presence of the HER2 fragment of sequence SEQ ID NO: 1 with monoclonal antibodies directed to neo-epitopes defined by SEQ ID NO: 2 or SEQ ID NO: 3.

Using the same peptide of SEQ ID NO: 4 of example 2, monoclonal antibodies were obtained. Of all of them, the monoclonal antibody 20F4 produced by the hybridoma cell line with registration number DSM ACC2904 and the monoclonal antibody 32H2 produced by the hybridoma cell line with registration number DSM ACC2980 were selected. The results obtained with this antibody are shown in Figure 5.

In a manner similar to that of Example 2, an SDS electrophoresis and subsequent Western-type transfer with a 1: 1: 1 mixture of MCF7 cell lysate extracts (previously transformed with the constructs of Figure 1) were carried out ), which expressed the complete form of the HER2 receptor, or the truncated form CTF-611, or the truncated form p95. In Figure 5A, which is a photograph of the Western membrane revealed with CB11 or with monoclonal antibodies 20F4 and 32H2, it can be seen that the latter specifically recognize the truncated form of HER2 corresponding to the CTF-611 receptor of SEQ ID NO: 1 and does not exhibit any detectable cross reactivity with the complete form of said receptor, which means that it actually recognizes a new epitope (neo-epitope) that includes the primary amino group at the N-terminal end of the CTF-611 form of the receptor .

Similarly, when an immunoprecipitation assay was performed with monoclonal antibodies 20F4 and 32H2 compared to CB11 antibody (which recognizes the cytosolic domain of receptors), it was detected that the monoclonal antibody directed against the SEQ peptide ID NO: 3 precipitated exclusively the truncated form of the receptor (CTF-611). These results are evident in Figure 5B. In this figure, the results of an electrophoresis with SDS and the subsequent Western-type transfer are shown, in whose lanes the results of immunoprecipitation tests with antibodies 20F4, 32H2 and CB11 were loaded.

In the lane corresponding to immunoprecipitation with antibodies 20F4 and 32H2, only the bands of the glycosylated and non-glycosylated form can be distinguished from the truncated form CTF-611. That is, antibodies 20F4 and 32H2 do not immunoprecipitate the complete form of the HER2 receptor. Therefore, it can be concluded that antibodies 20F4 and 32H2 are highly specific for CTF-611 and do not recognize the complete form of HER2, which means that they can be used for differential and highly selective detection of which form of the HER2 receptor is expressed in a isolated sample of tumor tissue.

The characterization of monoclonal antibodies directed against a peptide corresponding to the sequence, 32 amino acids in length, N-terminal, 611-CTF, confirms the existence of masked epitope (s) in full-length but exposed HER2 ) in 611-CTF. These epitopes are masked both in the solubilized molecule and in intact cells. The quantification of the different isoforms of HER2 in the immunoprecipitates CB11, 32H2 and 20F4 normalized with respect to the number of occurrences of HER2 is as follows:

CB11

32H2 20F4

HER2

one one one

611-CTF

1.01 343.50 1302.19

648-CTF

0.79 nd nd

Example 4: Characterization of a monoclonal antibody against the N-terminal end of 611-CTF by flow cytometry 5

(A) MCF77 cells expressing HER2, 611-CTF or 648-CTF were analyzed by flow cytometry using different concentrations of 32H2, a monoclonal antibody produced against the MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC peptide (see Figure 1). See figure 6 (A).

10 (B) The results of two independent experiments performed as in (A) were quantified and the averages are shown. See figure 6 (B).

(C) MCF7 cells expressing HER2, 611-CTF or 648-CTF were analyzed in a confocal microscope by indirect immunofluorescence with the indicated antibodies.

15 As a secondary antibody to fluorophore, goat anti-mouse IgG antibody Alexa Fluor 488 from Invitrogen (A110011) was used. To evaluate non-specific binding as a negative control, FACS was performed without 32H2 serum but in the presence of this secondary antibody ("2ary").

Conclusion: The epitope recognized by the 32H2 antibody is exposed in living cells that express 611-CTF but masked in living cells that express HER2.

Example 5: Characterization of monoclonal antibodies against the N-terminal end of 611-CTF by immunohistochemistry

25 Immunohistochemical staining of MCF7 cells expressing HER2, 611-CTF or 648-CTF was carried out with CB11, an antibody against the cytoplasmic domain of HER2, or with two independent anti-611-CTF monoclonal antibodies raised against the MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC peptide (DKGC). see figure 1). The result is shown in figure 7.

30 Conclusions. Epitopes recognized by antibodies 20F4 and 32H2 are exposed in cells expressing 611-CTF analyzed by immunohistochemistry. In contrast, evaluated with the same technique, these epitopes are masked in the full-length HER2 molecule. This result is particularly relevant due to the fact that immunohistochemistry is the technique of choice for most tests of

35 routine in clinical practice.

Example 6: Characterization of epitopes recognized by monoclonal antibodies against the Nterminal end of 611-CTF

40 (A) Scheme showing the main sequence of the juxtamembrane region of 611-CTF. The N and C-terminal ends of the molecule are indicated. The transmembrane and kinase domains are indicated by a striped box and a gray box, respectively. The sequence of the different deletion constructs is indicated. See figure 8 (A).

(B) Transiently transfected MCF-7 cells were lysed with cDNA deletion constructs starting at

45 the indicated amino acids. Cell lysates were analyzed by Western blotting with CB11, an antibody against the cytoplasmic domain of HER2, or with two independent anti-611-CTF monoclonal antibodies raised against the MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC peptide. See figure 8 (B).

Conclusions The epitopes recognized by the 32H2 and 20F4 antibodies are contained in or at least overlap with the MPIWKFPDEEC sequence.

Example 7: Analysis of human breast cancer samples with a monoclonal antibody against the Nterminal end of 611-CTF or with Herceptest ™

55 (A) In the indicated samples, HER2 expression was analyzed with Herceptest ™, fluorescent in situ hybridization (FISH) or Western blot. Those samples that expressed detectable levels of HER2 carboxyl terminal fragments (also known as P95) evaluated by Western blotting are indicated (see (Scaltriti, M., Rojo, F., Ocana, A., Anido, J., Guzman , M., Cortes, J., Di Cosimo, S., Matias-Guiu, X., Ramon y Cajal, S., Arribas, J., and Baselga, J. (2007) J Natl Cancer Inst 99 (8) , 628-638)). See figure

60 9 (A).

(B) Immunocytochemical staining of the same breast cancer samples as in A, with 32H2, an anti-611-CTF monoclonal antibody raised against the MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC peptide (see Figure 1) or with Herceptest ™, which stains the cytoplasmic domain of HER2. See figure 9 (B).

Conclusion. Using immunohistochemistry, the 32H2 antibody stains samples of human breast cancer that were previously known to express detectable levels of CTF, evaluated by Western blotting.

Example 8: Generation of animal models to characterize the effect of CTF expression in vivo

Mouse models have played a decisive role in showing the oncogenic potential and relevance of HER2 in tumor evolution. To characterize their oncogenic potential, transgenic mice (TG) expressing 611-CTF have been established under the control of the long terminal repeat of mouse mammary tumor virus, which is preferably active in the mammary gland. Although cellular models indicated that soluble intracellular CTFs are inactive, to further explore the consequences of the expression of these fragments, TG animals expressing 687-CTF were also generated. As a control, the classic and well characterized model that expresses natural type HER2 (ie rat neu) has been used.

At 7 weeks of age, the levels of 611-CTF expressed in lines F3 and F2 of heterozygous TG 611 were ay equal to and 1/3 of, respectively, the levels of endogenous HER2, although the level in F1 was below the threshold detection. The 687-CTF levels in the developed homozygous lines varied from  double to half of the HER2 levels in the F2 and F1 lines of TG 687, respectively.

The mammary glands of the TG animals did not show macroscopic abnormalities at 7 weeks of age. However, morphological examination of complete preparations stained with carmine revealed hyperplastic abnormalities in the breast ductal trees of TG HER2 mice. Similar, although less pronounced, abnormalities were present in the three lines of TG 611 mice. In contrast, the glands of TG 687 mice could not be distinguished from those of wild-type mice.

Example 9: Expression of 611-CTF leads to the development of aggressive breast tumors

Despite the more pronounced hyperplasia in TG HER2 mice, the three lines of TG 611 animals developed more aggressive tumors in terms of the number of tumors per animal, tumor growth and tumor appearance:

Mammary glands

Average mice (weeks) n

HER2 (Neu) 30.3 ± 7.5 22

611-F1 26.3 ± 4.6 6

611-F2 22.2 ± 4.8 12

611-F3 23.7 ± 5.5 3 (The appearance of breast tumors was monitored by weekly palpation.)

No tumors or abnormalities were observed in TG 687 animals even after a follow-up of more than one year.

Histological analysis of the tumors showed the same typical invasive solid nodular carcinomas induced by HER2 in TG 611 mice. The only histological difference between tumors initiated by HER2 and 611-CTF was a larger number of mitotic images in those of TG mice. 611

As shown above, TG HER2 mice developed pulmonary metastases. Three to six weeks after tumor detection,  1/4 of the TG HER2 animals had detectable nodules in the lungs:

Mice Pulmonary Metastasis n

HER2 (Neu) 22 9

611-F1, F2, F3 56 9 (Mice were sacrificed 3-6 weeks after tumor detection by palpation and metastasis was monitored by immunohistochemistry).

Histological analysis of pulmonary metastases confirmed the expression of HER2 and staining with cytokeratin 18 verified that the cells originated from the primary tumor. Compared to TG HER2, the number of animals expressing 611-CTF with detectable metastases was more than double. This shows that tumors initiated by this CTF have a more pronounced tendency to invade the lungs.

Sequences

SEQ ID NO: 1

SEQ ID NO: 2

SEQ ID NO: 3

SEQ ID NO: 4

SEQ ID NO: 5

5 Free text from the sequence list SEQ ID NO: 1 Truncated form or carboxyl terminal fragment (CTF) of the human HER2 protein SEQ ID NO: 2 Epitope of a truncated form of the human HER2 protein

SEQ ID NO: 3 Epitope of a truncated form of the human HER2 protein

SEQ ID NO: 4 Synthetic peptide derived from an epitope of a truncated form of the human HER2 protein 15 SEQ ID NO: 5 Synthetic peptide derived from an epitope of a truncated form of the human HER2 protein

Sequence list

<110> Private Foundation Institut de Recerca Vall Hebron University Hospital / Private Foundation Institut

20 d 'Oncology Research of Vall Hebron (VHIO) / Fundació Privada Catalan Institution of Research and Study Avançats

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<213> Homo sapiens

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40 <220>

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Claims (18)

1. Antibody or fragment thereof that recognizes an epitope of a truncated form of the HER2 receptor defined by SEQ ID NO: 1, said epitope being defined by a sequence included in SEQ ID NO: 2, in 5 that the antibody or fragment thereof is suitable for distinguishing the protein of SEQ ID NO: 1 from the HER2 receptor.

2.

Antibody or fragment thereof according to claim 1, wherein said epitope is one included in SEQ ID NO: 3.

3.

Antibody or fragment according to one or more of the preceding claims, wherein the antibody or fragment thereof is suitable for distinguishing the protein of SEQ ID NO: 1 from the truncated form 648CTF / p95 of the HER2 receptor.

Antibody or fragment according to claim 2 or 3, wherein the distinction can be visualized by one or more of immunofluorescence, flow cytometry, immunohistochemistry in cultured cells and immunohistochemistry in patient samples.

5.

Antibody or fragment thereof according to one or more of the preceding claims, which is a polyclonal antibody.

6.

Antibody or fragment thereof according to one or more of claims 1 to 4, which is a monoclonal antibody or a fragment thereof.

Antibody or fragment thereof according to claim 6, produced by the hybridoma cell line deposited in the "Deutsche Sammlung von Mikroorganismen und Zellen -DSMZ" with registration number DSM ACC2904 or the hybridoma cell line deposited in the "Deutsche Sammlung von Mikroorganismen und Zellen -DSMZ ”with registration number DSM ACC2980.

8.

Antibody or fragment according to one or more of the preceding claims, wherein the fragment is one selected from the group consisting of F (ab), F (ab ’) and Fv.

9.

Hybridoma producing the monoclonal antibody according to claim 6 or 7.

A peptide consisting of SEQ ID NO: 3 or SEQ ID NO: 4, which is optionally conjugated with an immunogen, for use in a method of producing an antibody according to one or more of claims 1 or 2, a method which involves immunization with said peptide.

eleven.

Method for obtaining a monoclonal antibody, wherein the hybridoma cell line according to claim 9 is grown in a suitable culture medium and the monoclonal antibody is recovered from this medium.

12.

Cancer diagnosis method, which comprises the detection of the presence of the truncated form of the HER2 receptor consisting of the amino acid sequence SEQ ID NO: 1 in a patient sample, in

45 that the detection is carried out with means selected independently of the group comprising:

(i)

detection through differential migration of the truncated form of the HER2 receptor consisting of SEQ ID NO: 1, in relation to a complete form of said HER2 receptor; Y

(ii)

detection by binding with one or more antibodies according to one or more of claims 1 to 11.

13. Method according to claim 12, which is a prognostic method that allows the prognosis of the evolution of tumor growth and / or metastasis.

14.

Use of an antibody or a fragment thereof according to any one of claims 1 to 8, for the diagnosis and determination of the prognosis in isolated samples of cancers in which the HER2 receptor is expressed.

fifteen.

Agent for the diagnosis and determination of the prognosis in isolated samples of cancers of the type expressing the HER2 receptor or its truncated variants, comprising at least one antibody or a fragment thereof according to any one of claims 1 to 8.

16. A diagnostic agent according to claim 15, wherein the antibody or fragment thereof is disposed on a solid support. 17. Kit for diagnosis and prognosis determination in isolated samples of cancers of the type expressing the HER2 receptor or its truncated variants, comprising the antibody or a fragment thereof according to any one of claims 1 to 8 to detect the presence in the truncated way 5 of the HER2 receiver consisting of SEQ ID NO: 1 in the sample. 18. Kit for diagnosis and prognosis determination according to claim 17, wherein the detection means comprise at least one agent according to claims 15 or 16. 19. A kit for the diagnosis and determination of the prognosis according to claim 17, wherein the detection means consists of the detection by differential migration of the truncated form of the HER2 receptor of SEQ ID NO: 1, in relation to the form complete of said HER2 receiver. 20. Antibody or fragment thereof according to one or more of claims 1 to 9, for therapeutic use. fifteen 21. Antibody or fragment according to claim 20, for use in the treatment or prevention of cancers in which at least the truncated form of the HER2 receptor consisting of SEQ ID NO is expressed: one. 22. Antibody or fragment according to claim 21, for use in the treatment or prevention of breast cancers in mammals, including humans. 23. Antibody or fragment according to one or more of claims 21, for use in the treatment or prevention of cancers selected from the group comprising lung, pancreas, colon, 25 stomach, prostate, head and neck, skin, kidney, testis, thyroid, urinary bladder, uterus, vulva, endometrium, ovary, esophagus, mouth, salivary gland, larynx, peritoneum, nasopharyngeal region, fallopian tubes, Wilms tumors, as well as lymphomas, Swing sarcomas, synovial sarcoma, medulloblastomas, trophoblastic tumors, gliomas, glioblastomas, cholangiocarcinomas, cholesteatoma, chondrosarcoma, ependymoma, neurilemomas, neuromas, rhabdomyosarcomas. 24. Use of an antibody or fragment thereof according to one or more of claims 1 to 8, for the manufacture of a medicament for the treatment of cancer in which at least the truncated form of the HER2 receptor consisting of SEQ ID is expressed. NO: 1. 35 25. Pharmaceutical composition for the treatment of cancers in which at least the truncated form of the HER2 receptor containing SEQ ID NO: 1 is characterized, characterized in that it comprises an antibody or a fragment thereof according to any one of claims 1 to 8 and at least one pharmaceutically acceptable excipient and / or carrier.